Transcript The Wild World of Biotechnology!! Applications Genetic

The Wild World of
Biotechnology!!
Applications
Genetic Transformation
Cloning - Genes and entire organisms
Gene Therapy
Environmental Clean-Up
How do we genetically
engineer or modify an
organism?
Selective breeding
Genetic transformation
Genetic Transformation
This is the process by which we get an organism to
express foreign DNA
e.g. making a tomato synthesize antifreeze proteins
that are commonly found in fish
e.g. making a bacterial cell synthesize human insulin
Recombinant DNA
Recombinant DNA = a molecule of DNA that contains
DNA from two or more organisms
Making recombinant DNA is the first step in producing
genetically engineered (GE) or genetically modified
(GM) organisms
Making Recombinant
DNA
Isolate the gene of interest (e.g. the gene for insulin)
using restriction enzymes
This is a hit or miss process that requires a great deal
of luck to be successful
Restriction enzymes generally cut at palindromic DNA
sequences
Making Recombinant
DNA continued...
Some method is needed to get the gene of interest
into the cells of the organism we wish to transform
e.g. plasmid vectors and gold particles or
electroportation
Polymerase Chain
Reaction (PCR)
Another DNA cloning technique
Using plasmid vectors
These are small circular bits of DNA that are separate
from the main prokaryote chromosome.
They are easily transformed because of there
chemistry
Bacterial cells will absorb foreign plasmids and
express their genes. You and I cannot do this!!!
pGLO
pGLO are plasmids that carry the genes for
fluorescent proteins from jellyfish
Getting bacteria to
express pGLO genes
The trick is getting the recombinant plasmid past the cell membrane
and into the cell and then getting the cell to express the genes.
We use chemicals (CaCl2) and heat shock to get recombinant
plasmids into the cell.
We include antibiotic resistance genes in the recombinant plasmid
so that only the successfully transformed bacteria live.
We make sure the gene of interest is near a known operon and we
intentionally turn that operon on (e.g. arabinose, tryptophan,
lactose)
Gene cloning
Once the bacteria take up the recombinant plasmid
they will go through DNA replication and, in effect,
clone the gene of interest many times
Studying DNA
Gel electrophoresis
A method for separating nucleic acids based on size
and electrical charge
Gel Electrophoresis
A DNA sample is cut with restriction enzymes.
The cut up DNA is placed in one end of a gel and
electricity is passed through the gel
Because DNA carries a negative charge the electric
current is able to carry the DNA through the gel
The smallest pieces of DNA move the furthest
distance
RFLPs
Restriction fragment length polymorphisms
This term refers to variation in a gene sequence in a
population
RFLPs can be used to determine paternity, in
forensics, to map genes, and to detect alleles
Draw a restriction map of
the plasmid...